Aurelia: Jurnal Penelitian dan Pengabdian Masyarakat Indonesia System Analysis of Employee Performance and Facility Supervision by the Terminal Inspection Service Unit at Tjilik Riwut Palangkaraya Airport
July 2023

Erik Tirtajaya email@example.com
Andityo Pujo Laksana
Aurelia: Jurnal Penelitian dan Pengabdian Masyarakat Indonesia System Analysis of Employee Performance and Facility Supervision by the Terminal Inspection Service Unit at Tjilik Riwut Palangkaraya Airport
22July 2023Air Transportation Management Study ProgramSekolah Tinggi Teknologi Kedirgantaraan YogyakartaBantul RegencyProvince of Daerah Istimewa YogyakartaIndonesia 12 Keywords: Employee performance systemStandard Operating ProceduresTerminal Inspection Service (TIS)
Supervision of standard facilities at Palangkaraya Airport is the duty and responsibility of the Terminal Inpetion Service (TIS). The most important thing for the smooth operation of the Terminal Inspection Service (TIS) on the land side is the employee's performance in monitoring facilities with existing resources so that supervision of facilities on the land side can be carried out optimally. The purpose of this study is to determine the performance system of Terminal Inspection Service (TIS) employees for monitoring facilities at Tjiik Riwut Palangkaraya Airport. This research uses descriptive qualitative research method, the data that has been obtained will be analyzed qualitatively and described in a descriptive form. The data used in this research are primary data and secondary data. Primary data in this study included interviews with Terminal Inspection Service (TIS) unit personnel, while secondary data were obtained from Terminal Inspection Service (TIS) documents and literature. Based on the research results it is known that in carrying out their duties and responsibilities, the Terminal Inspection Service (TIS) unit still lacks personnel. Tasks that are still not handled optimally, namely supervision on the land side (Landside) namely, monitoring of Trolley facilities and air temperature in the terminal waiting room section, Trolleys are often scattered (out of place) and passengers often complain of overheating, due to the number of personnel who insufficient so that supervision of landside facilities cannot be maximized.

INTRODUCTION

The air transportation system in Indonesia is increasingly playing a role in economic development and is the authority of air transportation to be able to serve the entire archipelago, especially in relation to accelerating the flow of information, goods, passengers and so on. Air transportation is very important because areas that were previously difficult to reach by land transportation can now be reached through air transportation to connect in the fields of economy, government, tourism and others. The airport is a supporting infrastructure for air transportation, where airplanes can take off and land. To support the comfort of airport passengers, there are several requirements that must be met by airport managers, one of which is adequate and functioning facilities. To ensure that these facilities function properly, these facilities need to be supervised. Tjilik Riwut Airport is an airport managed by the state-owned company PT Angkasa Pura II Tjilik Riwut Airport, Palangka Raya, formerly known as Panarung Airport, is the largest airport in Palangka Raya, Central Kalimantan. Providing a good image is one of the keys to success for aviation services at Tjilik Riwut Airport.

Performance is a condition that must be known and confirmed to certain parties to determine the level of achievement of an agency's results in relation to the vision carried out by an organization or company and to know the positive and negative impacts of an operational policy. Performance according to Anwar Prabu Mangkunegara (2000: 67). Performance (work performance) is the result of work in quality and quantity achieved by an employee in carrying out his duties in accordance with the responsibilities given to him. Supervision is all activities to ensure and guarantee that the tasks/work have been carried out in accordance with the plans that have been set, the policies that have been outlined and the orders (rules) given. Supervision in an organization is needed if the organization will achieve organizational goals. The surveillance security system is a way of observing, monitoring and identifying certain objects, this is done so that each activity can run according to the set goals, if there are problems it is immediately resolved. The airport facility monitoring system carried out by every airport officer nationally refers to Regulation of the Minister of Transportation Number 178 of 2015 concerning Service Standards for Airport Service Users.

Supervision of facilities at airports is based on the Zero Accident principle. The Zero Accident Award or zero accident award is one of the awards given by the Ministry of Manpower of the Republic of Indonesia to companies that have succeeded in implementing work safety and health programs so as to achieve zero work accidents at a certain time. namely to overcome the possibility of incidents of accidents at the work site, both minor accidents and fatal accidents that result in death. Therefore it is important to carry out supervision at each airport facility to ensure that the facilities used by passengers are ready and ready to be used.

Terminal Inspection Service (TIS) is a unit of PT. Angkasa Pura II, which is tasked with supervising and ensuring that the facilities at the terminal (airport) function properly, the supervision carried out refers to PM 178 of 2015 concerning Service Standards for Airport Service Users and SOP for the Terminal Inspection Service unit. At Tjilik Riwut Palangkaraya airport In their daily lives, TIS employees check facilities at the airport terminal related to passenger service and during busy times the number of passengers increases so that service cannot be maximized. There are still a number of things that need to be improved and paid attention to or added to from the terminal facilities at Tjilik Riwut Airport. In public areas (arrivals and departures) there are not enough signs for directions to the smoking room so that many airport service users smoke in the departure and arrival halls, there is often a shortage of trolly in the arrival area, prayer room signs in public areas, charger stations in the waiting room still use two legs, and the air conditioner in the waiting room is not cool enough.

The objectives of the research are: To find out the performance system of Terminal Inspection Service unit employees in monitoring airport facility security at PT.Angkasa Pura II (Persero) Tjilik Riwut Branch Office. What Are the Constraints of the Terminal Inspection Service Unit (TIS) When Supervising Airport Facilities.

Theoretical Basis Airport

According to the International Civil Aviation Organization, airports are defined areas on land and water (including buildings, installations and equipment) which are designated, either wholly or partly, for the arrival, departure and movement of aircraft. Meanwhile, according to PT (Persero) Angkasa Pura, an airport is an airfield, including all buildings and equipment which is a minimum to ensure the availability of facilities for air transportation for the public. According to the Law of the Republic of Indonesia Number 1 of 2009 concerning flights chapter 1 article 1: 1. Airport is everything related to the operation of airports and other activities in carrying out the functions of safety, security, smoothness and orderliness of the flow of aircraft, passenger, cargo and/or postal traffic, intra and/or intermodal movement places and increasing growth. national and regional economy. 2. The national airport order is the national airport system which describes airport planning based on spatial planning, economic growth, regional comparative advantage, natural and geographical conditions, integration of intra and intermodal transportation, environmental sustainability, aviation safety and security, as well as integration with other development sectors. 3. Airport is an area on land and/or waters with certain boundaries that is used as a place for aircraft to land and take off, take off passengers, load and unload goods, and place for intra and intermodal transportation, which will be equipped with safety and security facilities. flight safety, as well as basic facilities and other supporting facilities.

Terminal

Terminal is a base for public motorized vehicles used to manage arrivals and departures, loading and unloading people and/or goods, as well as changing modes of transportation. One component of the transportation system which has the main function as a temporary stop for public vehicles to raise and lower passengers and goods to the final destination of a trip, as well as a place for controlling, supervising, regulating and operating the system for the flow of passenger and goods transport, as well as functions to expedite the flow of passenger or goods transportation (PM 132 of 2015 chapter I article I).

Standard Operating Procedures

SOP, or the abbreviation for Standard Operating Procedures, is a series of standards that support well-organized work procedures in an organization or company. Standard Operating Procedure (SOP) is a guide used to ensure the operational activities of an organization or company run smoothly (Sailendra, 2015: 11). According to Tjipto Atmoko (2011), Standard Operating Procedures (SOP) is a guideline or reference for carrying out work tasks in accordance with the functions and performance appraisal tools of government agencies based on technical, administrative and procedural indicators according to work procedures, work procedures and work systems in the work unit concerned. Standard Operating Procedures (SOP) are needed by companies to produce quality systems, consistent technical, and maintain quality control and keep business processes running well. This SOP is usually related to products, human resources, and so forth. One of the company's SOPs that is very important is the Company's SOP regarding HR. SOP regarding HR is how the company manages the system of recruiting, training, signing contracts, issuing warning letters (SP), bonuses, and work leave.

Terminal Inspection

According to PM 178 of 2015 regarding service user standards (level of service) aimed at specific passengers at the terminal, namely, terminal inspection is to carry out supervision and inspection of services and facilities related to services provided to passengers. Terminal inspection operational system activities include providing instructions and supervision, checking and handling of all facilities, and to every operating personnel in the airport terminal area. Terminal Inspection is a unit under the Airport Service section that is very concerned about the security, comfort, smooth running and safety of anyone at the airport. SOP for Terminal Inspection is to supervise and make observations of facilities that support operations from entering the parking lot to passengers going to the terminal and boarding the plane. The supervision referred to in this case is coordinating regarding the existence of something that is not optimal in the provision of services, namely especially at the existing facilities at the terminal (Regulation of the Minister of Transportation of the Republic of Indonesia Number PM 178 of 2015 Article 3). According to PT Angkasa Pura I, a terminal inspection is a person who works as a TIS (Terminal Inspection Service) who has to go around the airport to check the condition of the airport. If something is broken, Terminal inspection will report to the expert who will fix it. Meanwhile, according to PT Angkasa Pura II Terminal Inspection is to ensure that the existing facilities at the terminal are functioning properly, if they are damaged or not functioning they will be followed up immediately. Inspection translated into Indonesian from English is an inspection according to KBBI (Big Indonesian Dictionary), inspection is a thorough examination; direct inspection of the implementation of regulations, duties. In quality control, inspection is a very important element. Inspection (inspection) is necessary to ensure the quality of products produced in accordance with the provisions and standards so that customer satisfaction can be maintained properly. According to Salma (2008) inspection is an examination carried out by direct observation. We apply the word Inspection or Inspection to quality control, so it can be interpreted that Inspection or Inspection is a careful examination of a product that is produced whether it is in accordance with the standards and rules that have been set for it.

Performance

According to Anwar Prabu Mangkunegara (2009:67) performance is the result of work in quality and quantity achieved by an employee in carrying out his duties in accordance with the responsibilities given to him. According to Soekidjo Notoatmodjo (2009: 124) argues that: performance is what a person can do according to his duties and functions. Factors that affect performance are the high and low performance of an employee of course determined by factors that influence either directly or indirectly . According to Anwar Prabu Mangkunegara (2009:67) states that: factors that affect performance achievement are ability factors and motivational factors. So from this it can be concluded that the factors that affect performance can come from within the individual employee or outside the individual. According to Sedarmayanti (2011: 260) performance is a translation of performance which means the work of a worker, a management process or an organization as a whole, where the work results must be shown concretely and can be measured (compared to predetermined standards).

Surveillance System

The surveillance security system is a way of observing, monitoring and identifying certain objects, this is done so that each activity can run according to the set goals, if there are problems, they are immediately resolved. The airport facility monitoring system carried out by every airport officer nationally refers to the Minister of Transportation Regulation Number 178 of 2015 concerning Airport Service User Service Standards. According to the concept of management, according to John R. Schermerchon, Jr. (1996:4), "Management is the process of planning, organizing, leading and controlling the use of resources to accomplish performance goals", which implies the following: Management is the process of planning , organizing, leading, and supervising/controlling the use of resources to achieve goals.

Facilities

According to Yazid (2013: 136) is part of the physical evidence of services. Here it is stated that physical evidence of services covers all aspects of the organization's physical facilities or the servicescape (including: created environment, man-made, physical environment). Meanwhile, according to Tjiptono (2006: 144) facilities are part of physical evidence, in this book it is explained in outline, physical evidence includes organizational physical facilities (Servicecape) and other forms of physical communication. According to Lupioadi (2008) regarding the definition of facilities is a means to expedite and facilitate the implementation of To find out the duties of the inspector terminal and must be in accordance with the SOP so that work is created in accordance with the conditions at the airport.

Source: Previous Research Data

RESEARCH METHODS

The research to be carried out is to use research methods with qualitative research methods. Data collection techniques carried out by triangulation (combination of observations, interviews, documentation). This activity and research was carried out at Palangka Raya Tjilik Riwut Airport from August 1 to September 30 2022. Tjilik Riwut Palangka Raya Airport is located Jl. Adonis Samad, Panarung, Kec. Pahandut, City of Palangka Raya, Central Kalimantan. In this research, the sources and types of data used are primary data and secondary data. 1. Primary Data. Primary data is data that is directly obtained from research objects related to research and is the main data. As for examples of primary data such as data from interviews. 2. Secondary Data. Secondary data is a source of indirect data obtained on the object of research. This secondary data was obtained from literature studies and other information related to research. 1. Interview. Yusuf, (2014:372). An interview is an event or process of interaction between the interviewer and the source of information or the person being interviewed through direct communication or asking directly about an object under study. a. Structured interviews: Structured interviews are used as a data collection technique, if the researcher or data collector knows for certain what information will be obtained. Therefore, in conducting interviews, data collectors have prepared a research instrument in the form of written questions. With this structured interview, each respondent was asked the same questions, and the data collectors recorded them. b. Unstructured interviews: These are free interviews in which the researcher does not use interview guidelines that have been arranged systematically and completely for the data collectors. The interview guide used is only an outline of the problems to be asked. The types of interviews conducted by the researchers in this study were structured and unstructured interviews. 2. Observation. This technique is the observation of research on the research object. The researcher made direct observations of the Terminal Inspection Service unit in implementing the Standard Operating Procedures for the supervision of the Landside Facility. Observation is a data collection technique that has specific characteristics when compared to other techniques. Observation is also not limited to people, but also other natural objects. Through observation activities researchers can learn about behavior and the meaning of this behavior. The observation in this study is by making direct observations in the field to find out the actual conditions at Tjilik Riwut Airport in Palangka Raya. This observation was carried out by direct observation of field conditions during the Work Practice. Such as the lack of checking on facilities to support the convenience of airport service users, for example: ac, trolly, charger station (triangle and USB), and directions (public area smoking area and prayer room). 3. Documentation. Documentation Yusuf, (2014) Documentation comes from the word document, which means written goods, the documentation method means procedures for collecting data by recording existing data. The documentation method is a data collection method used to trace historical data. Documents about people or groups of people, events, or occurrences in social situations that are very useful in qualitative research. This study uses data originating from the company in the form of SOP (Standard Operating Procedures), photos or pictures of the facilities at Tjilik Riwut Airport in Palangka Raya.

Data Collection Technique

RESEARCH RESULTS AND DISCUSSION TIS Unit Employee Performance System TIS Unit personnel

Based on interviews from informants: Ryano Harpanda, as (TIS Supervisor at Palangkaraya Airport) said that: There are only 3 personnel available in the Terminal Inspection Service (TIS) unit plus one Assistant Manager to 4 personnel, but the Assistant Manager does not go down directly to the field so that only 3 people counted. The availability of 3 personnel is still insufficient because there are many tasks that must be carried out by the personnel of the Terminal Inspection Service (TIS), plus if there are personnel who are on leave or sick, then no one backs up. So with a minimal number of personnel, it is felt that they are still lacking to carry out tasks in the Terminal Inspection Service (TIS).

With 2 personnel every 2 days, basically everything can be done, but because of the pressure with 2 people every day it will be very heavy because the duties and responsibilities of the Terminal Inspection Service (TIS) are very large, starting from the level of service including; Departure and arrival processes, convenience, and value-added facilities. Checking facilities according to Standard Operating Procedures, taking into account baggage starting from block on baggage being unloaded until it is picked up by airport service users, checking light on the land side (lindside), and so on. If there are only 2 people per day, the employee's performance in monitoring facilities cannot be maximized.

With the number of existing and still very few employees affecting the performance system of Terminal Inspection Service (TIS) employees at Palangkaraya Airport, there are several tasks that are still not optimal on the land side, the task in question is supervision of facilities, such as those Tjilik Riwut Palangkaraya airport in the landside terminal especially in Lack of time and a large number of passengers during rush hour means that the trolleys are usually scattered in public areas, lack of cleanliness in the waiting room, and the temperature is not according to procedures. The work that takes up a lot of employee time and requires more manpower is to calculate the Level Of Service (LOS), which is carried out 1 time for 1 month, while all reporting on the calculation of the Level Of Service every day is entered into the E-Service application, so that Trolley monitoring and reduced air temperature which resulted in Trolley scattered in public areas and many passengers complained of overheating.

Observation Results at the Terminal Inspection Service (TIS) Unit

The results of observations in this study refer to what was done by the Terminal Inspection Service (TIS) unit at Tjilik Riwut Palangkaraya Airport. Observations in this study are primary data obtained directly during field practice at Tjilik Riwut Palangkaraya Airport, which is primary data that supports research conducted at the Terminal Inspection Service (TIS) unit at Tjilik Riwut Palangkaraya Airport. The results of observations at the Terminal Inspection Service (TIS) unit are as follows:

& Andityo Pujo Laksana -Sekolah Tinggi Teknologi Kedirgantaraan Yogyakarta 1302

Erik Tirtajaya & Andityo Pujo Laksana -Sekolah Tinggi Teknologi Kedirgantaraan Yogyakarta 1300Aurelia: Jurnal Penelitian dan Pengabdian Masyarakat Indonesia
E-ISSN: 2964-2493 P-ISSN: 2962-0430
Vol. 2 No. 2 July 2023

functions and facilities are the appearance, capabilities of facilities and infrastructure and the condition of the surrounding environment in showing their existence to externals which includes physical facilities (buildings) equipment and tools. According to Sam: 2012, facilities are a means to expedite the implementation of a particular business. From this elaboration we can understand that anything that can expedite work or effort to achieve a goal can be referred to as a facility.Aurelia: Jurnal Penelitian dan Pengabdian Masyarakat Indonesia
E-ISSN: 2964-2493 P-ISSN: 2962-0430
Vol. 2 No. 2 July 2023

Erik Tirtajaya & Andityo Pujo Laksana -Sekolah Tinggi Teknologi Kedirgantaraan
Yogyakarta
1301

Relevant Research

Table 1. Relevant Research
No
Name
Titel
Year
Research Result

1
Harry P.
Situmorang

The Role and Strategy of
Terminal Inspection in
supporting the land side
services of Yogyakarta
Adisutjipto International
Airport

2016
To create good service for passengers so as
to lead to passenger satisfaction.

2

Budiyarti
Eko
Setyaningsih

The effect of service quality on
airplane passenger satisfaction
at Adisoemarmo International
Airport, Surakarta

2010

The quality of service or the results of the
performance of each officer at the airport
plays an important role, especially for
passenger satisfaction and influences each
other.

3
Mohammad
Iqbal

Yogyakarta Adisutjipto
International Airport inspector
terminal

2015

Erik Tirtajaya & Andityo Pujo Laksana -Sekolah Tinggi Teknologi Kedirgantaraan Yogyakarta 1303 the waiting room and also the air temperature does not meet the applicable standards. so many passengers complain of overheating. During peak hours, Terminal Inspection Service (TIS) employees usually check the facilities on the land side of Tjilik Riwut Airport, Palangkaraya, because the number of TIS employees is only 2 people per day, consisting of Supervisors TIS and TIS Offcier, TIS Supervisor monitors if there is damage or information on lack of cleanliness at the terminal and makes daily reports if there is damage to existing facilities at the landside terminal, while the TIS officer checks the existing facilities at Level Of Service (LOS) if there are findings of damage and lack of cleanliness then it is immediately reported to the TIS Supervisor.Aurelia: Jurnal Penelitian dan Pengabdian Masyarakat Indonesia
E-ISSN: 2964-2493 P-ISSN: 2962-0430
Vol. 2 No. 2 July 2023

Table 2 .
2Observation of the Tjilik Riwut Facility It's over, because the arrival lane is wide, clean and also comfortable and there are no complaints from prospective passengers or passengers a. Broad vehicle arrival lanes √ b. Clean √ c. Comfortable √ 2. Departure and arrival trolley facilities are available to help make it easier for passengers Appropriate, because it makes it easier for passengers to carry lots of heavy goods 3. There is a self check-in facility. √No Indicator
Supervision Aspect (20
September-30 September 2022)

in
accordance

it is not in
accordance
Findings

1

Departure
and Arrival
Process

1. Vehicle departure lane
facilities:

√

2

Comfort
and
Cleanliness

1. Departure waiting room
facilities provided by the Tjilik
Riwut Palangkaraya Airport
terminal

Inappropriate,
departure lounge
facilities are spacious
and clean but not
comfortable due to the
hot air temperature so
many passengers
complain of
overheating.

a. Wide
√
b. Clean
√
√

c. Comfortable

2. Air-conditioning facilities at the
Tjilik Riwut Palangkaraya
airport terminal

Not suitable, the air-
conditioning facilities
are clean, but not cool

TIS Unit Supervision ScopeSupervise airport facilities functioning properly in accordance with the applicable Standard Operating Procedures (SOP) which refers to PM 178 of 2015 concerning Airport Service User Service Standards. Includes: 1. Check service standards and ensure airport facilities are functioning normally. 2. Ensure that the cleanliness of the airport meets the company's standards. 3. Checking the Level Of Service (LOS) in accordance with the standard provisions set in PM 178 of 2015. 4. Checking the Trolley facilities during peak hours so as not to reduce the trolley facilities obtained by passengers.The performance of Terminal Inspection Service (TIS) unit officers at Tjilik Riwut Palangkaraya Airport in supervising landside terminal facilities is in accordance with the SOP. but there are some that are still not, due to adjusting the number of passengers there is electricity savings for facilities that use electricity so that waste does not occur. When carrying out their duties, Terminal Inspection Service (TIS) employees are also equipped with supporting facilities such as TIS vests and HT. In order to facilitate TIS's work in communicating both to passengers who want to ask questions about facilities and to communicate with parties who work with TIS, if something happens in the terminal such as a problem with electricity, the TIS officer will immediately contact the electricity officer, and also if there is a problem in the conveyor upon arrival, the TIS officer immediately contacted the technician, what often happened was usually the cleaning department both in the waiting room and the toilet, so the tis officer immediately contacted the cleaning party to immediately follow up.CONCLUSIONThe performance of Terminal Inspection Service (TIS) unit employees at Tjilik Riwut Palangkaraya Airport is still lacking. Because the number of employees is only 3 personnel divided into 2 people every day basically everything can be done, but because the pressure that is obtained with 2 people per day is very heavy due to the duties and responsibilities of the Terminal Inspection Service (TIS) unit for existing facilities at Tjilik Airport Riwut Palangkaraya, especially on the land side, starting from checking facilities to cleanliness at the terminal, must comply with the applicable Standard Operating Procedures (SOP) referring to PM 178 of 2015 Airport Service User Service Standards.In carrying out the duties, responsibilities and authorities of the Terminal Inspection Service (TIS) unit, it has been divided equally according to the capabilities of each, so that in carrying out their duties the personnel of the Terminal Inspection Service (TIS) unit can work together in coordination with other employees. Tasks that are still not handled optimally at this time, namely, supervision of trolleys that are often scattered (not in the right place), because Terminal Inspection Service (TIS) officers focus on Level Of Service (LOS), so supervision of Trolleys is not optimal.From the results of the research, discussion, and conclusions that have been described, the researcher can provide several suggestions aimed at building and improving the implementation of tasks so that they are in accordance with those in the Standard Operating Procedures (SOP), including the following: To the Team Leader, make a letter of submission for additions personnel in the Terminal Inspection Service (TIS) unit if it is absolutely necessary so that the tasks can be carried out properly in accordance with the Terminal Inspection Service (TIS) procedures. It is hoped that employees of the Terminal Inspection Service (TIS) unit will be able to carry out their respective duties and responsibilities and be able to improve performance on facility supervision in accordance with Standard Operating Procedures (SOP).BIBLIOGRAPHY
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CELLULAR AND INFECTION MICROBIOLOGY ORIGINAL RESEARCH ARTICLE Phase variable expression of capsular polysaccharide modifications allows Campylobacter jejuni to avoid bacteriophage infection in chickens
published: 20 February 2012

Martine C Holst Sørensen
Department of Veterinary Disease Biology
University of Copenhagen
FrederiksbergDenmark

†
Lieke B Van Alphen
Department of Biological Sciences
Alberta Glycomics Centre
University of Alberta
EdmontonABCanada

†
Christopher Fodor
Department of Biological Sciences
Alberta Glycomics Centre
University of Alberta
EdmontonABCanada

Shauna M Crowley
Department of Biological Sciences
Alberta Glycomics Centre
University of Alberta
EdmontonABCanada

Bjarke Bak Christensen
Ottawa Institute of Systems Biology
National Food Institute
Technical University of Denmark
SøborgDenmark, Canada

The University of Tennessee
USA

Department of Biological Sciences
Alberta Glycomics Centre
University of Cambridge
UK

Department of Veterinary Disease Biology
University of Alberta
T6G, 2E9EdmontonABCanada

University of Copenhagen
Stigbøjlen 4DK-1879Frederiksberg CDenmark

Christine M Szymanski christine.szymanski@
Department of Biological Sciences
Alberta Glycomics Centre
University of Alberta
EdmontonABCanada

Lone Brøndsted
Department of Veterinary Disease Biology
University of Copenhagen
FrederiksbergDenmark

Alain Stintzi
Jun Lin
Duncan Maskell
Christine M Szymanski
CELLULAR AND INFECTION MICROBIOLOGY ORIGINAL RESEARCH ARTICLE Phase variable expression of capsular polysaccharide modifications allows Campylobacter jejuni to avoid bacteriophage infection in chickens
published: 20 February 201210.3389/fcimb.2012.00011Edited by: Reviewed by: *Correspondence:bacteriophageCampylobacter jejunicapsular polysaccharidephase variationphosphoramidatemethylation
† Martine C. Holst Sørensen and Lieke B. van Alphen have contributed equally to this work.Bacteriophages are estimated to be the most abundant entities on earth and can be found in every niche where their bacterial hosts reside.The initial interaction between phages and Campylobacter jejuni, a common colonizer of poultry intestines and a major source of foodborne bacterial gastroenteritis in humans, is not well understood. Recently, we isolated and characterized a phage F336 resistant variant of C. jejuni NCTC11168 called 11168R. Comparisons of 11168R with the wildtype lead to the identification of a novel phage receptor, the phase variable O-methyl phosphoramidate (MeOPN) moiety of the C. jejuni capsular polysaccharide (CPS). In this study we demonstrate that the 11168R strain has gained cross-resistance to four other phages in our collection (F198, F287, F303, and F326). The reduced plaquing efficiencies suggested that MeOPN is recognized as a receptor by several phages infecting C. jejuni. To further explore the role of CPS modifications in C. jejuni phage recognition and infectivity, we tested the ability of F198, F287, F303, F326, and F336 to infect different CPS variants of NCTC11168, including defined CPS mutants. These strains were characterized by high-resolution magic angle spinning NMR spectroscopy. We found that in addition to MeOPN, the phase variable 3-O-Me and 6-O-Me groups of the NCTC11168 CPS structure may influence the plaquing efficiencies of the phages. Furthermore, co-infection of chickens with both C. jejuni NCTC11168 and phage F336 resulted in selection of resistant C. jejuni bacteria, which either lack MeOPN or gain 6-O-Me groups on their surface, demonstrating that resistance can be acquired in vivo. In summary, we have shown that phase variable CPS structures modulate phage infectivity in C. jejuni and suggest that the constant phage predation in the avian gut selects for changes in these structures leading to a continuing phage-host co-evolution.

INTRODUCTION

Bacteriophages (phages) are the most abundant group of organisms on the planet and are estimated to outnumber their bacterial hosts by 10-fold (Brüssow and Hendrix, 2002). The first step of phage infection is through the specific recognition and attachment of the phage particle to the host cell (Weinbauer, 2004). In general, phages bind to unique host-specific structures, thus allowing them to recognize a suitable host in a mixed bacterial population (Rakhuba et al., 2010). Knowledge of phage receptors is required for implementation of efficient phage therapies against human pathogens -an intervention strategy that has been Abbreviations: β-D-Rib, ribose; β-D-GalfNAc, N -acetylgalactosamine in the furanose configuration; α-d-GlcpA6(NGro), glucuronic acid with 2-amino-2deoxyglycerol at C-6; MeOPN, O-methyl phosphoramidate; Hepp, heptose. The 2-amino-2-deoxyglycerol can also be substituted with an N -ethanolamine modification at C-6. promoted by the increasing occurrence of antibiotic resistance (Levy and Marshall, 2004;Kutateladze and Adamia, 2010), yet only a few bacterial phage receptors have been identified in Gramnegative bacteria (for example, Gemski and Stocker, 1967;Stirm and Freund-Molbert, 1971;Gemski et al., 1975;Yu and Mizushima, 1982;Morona et al., 1984).

Capsular polysaccharides (CPS) are highly hydrated polymeric gels that provide a thick (400 nm or more) layer protecting bacteria from hostile environments and host immune defenses, but the CPS can also be a target for phage binding and infection (Stirm and Freund-Molbert, 1971;Roberts, 1996;Comstock and Kasper, 2006). The capsule of the zoonotic foodborne pathogen, Campylobacter jejuni, is required for serum resistance, invasion of human epithelial cells, colonization of chickens, and diarrheal disease in ferrets (Bacon et al., 2001;Jones et al., 2004). Furthermore, mutation of several C. jejuni CPS genes resulted in phage resistance against a group of phages, but the actual capsular moieties involved Frontiers in Cellular and Infection Microbiology www.frontiersin.org

were not identified (Coward et al., 2006). Only recently a specific component of the C. jejuni capsule was identified as the phage receptor for this species. We showed that phage F336 relies on the O-methyl phosphoramidate (MeOPN) modification of the C. jejuni NCTC11168 CPS for adsorption to the host cell surface and infection (Sørensen et al., 2011).

The CPS of C. jejuni is the major component recognized by the Penner serotyping scheme for which 47 different serotypes are known (Guerry et al., 2012). Thus, the CPS are highly diverse in structure and this is reflected by the gene content of the capsular loci in various C. jejuni strains (Karlyshev et al., 2005). In addition, a number of CPS genes possess homopolymeric tracts making them prone to phase variation which allows a rapid on/off switching of the genes and promotes variations in CPS even when the strains have identical gene contents (Linton et al., 2001;St Michael et al., 2002;Karlyshev et al., 2005;McNally et al., 2005;Chen et al., 2008). (St Michael et al., 2002;Szymanski et al., 2003;McNally et al., 2005). The CPS biosynthetic locus in strain NCTC11168 includes 28 genes and 6 of these genes (cj1420, cj1421, cj1422, cj1426, cj1429, and cj1437 ) contain homopolymeric G (polyG) tracts (Parkhill et al., 2000). Even though the function of only three out of the six gene products has been identified, a highly variable presence/absence of methyl, ethanolamine, aminoglycerol, and phosphoramidate modifications of the CPS have been observed (St Michael et al., 2002;Szymanski et al., 2003;McNally et al., 2005), suggesting that the remaining genes could encode enzymes involved in synthesis or transfer of these modifications to the CPS glycans. Recently, the cj1421 and cj1422 genes were shown to encode MeOPN transferases attaching MeOPN to the GalfNAc and the Hep residues, respectively (McNally et al., 2007). Similarly, cj1426 was demonstrated to encode the 6-O-Me transferase enzyme (Sternberg et al., submitted).
The CPS of C. jejuni 11168 consists of -2)-β-d-Ribf-(1-5)-3-MeOPN-β-d-GalfNAc-(1- 4)-α-d-GlcpA6(N Gro)-(1-backbone with a 3,6-di-O-methyl-4- MeOPN-d-glycero-α-l-gluco-Hepp side chain
We previously discovered that resistance toward phage F336 evolved with a high frequency in C. jejuni NCTC11168 due to loss of the MeOPN receptor on the bacterial surface as a result of phase variation in the cj1421 gene encoding the MeOPN-GalfNAc transferase. In addition, deletion of the cj1421 gene in NCTC11168 resulted in phage resistance, proving that MeOPN attached to GalfNAc was a receptor for phage F336 (Sørensen et al., 2011). The aim of the present study was to determine if other phages rely on the MeOPN moiety for infection of C. jejuni and to explore the role of other CPS phase variable modifications for phage infection. Knowledge of phage receptors and development of phage resistance in vivo is essential for implementation of efficient phage therapy against C. jejuni in the chicken gut. We found that not only the MeOPN moiety, but also the phase variable 3-O-Me and 6-O-Me groups in the CPS of C. jejuni NCTC11168 may influence phage sensitivity. Co-infection of chickens with C. jejuni NCTC11168 and phage F336 resulted in bacterial acquisition of phage resistance through a loss of MeOPN attached to GalfNAc of the capsule or the acquisition of the 6-O-Me group. We propose that the constant exposure of C. jejuni to naturally occurring phages in the avian gut selects for different phase variable structures of the CPS resulting in a continuous phage-host co-evolution.

MATERIALS AND METHODS

BACTERIAL STRAINS, MEDIA, AND GROWTH CONDITIONS

The C. jejuni strains used in this study are listed in Table 1 and were routinely grown on blood agar Base II (Oxoid) supplemented   Sails et al. (1998). b HhaI restriction pattern as determined in Hansen et al. (2007) (Hansen et al., 2007). Stock bacteriophages were kept in SM buffer (0.05 M Tris-Cl, pH 7.5 supplemented with 5.8 g NaCl, 2.0 g MgSO 4 ·7H 2 O, and 5 ml gelatin, 2% w/v solution) at 4˚C.

BACTERIOPHAGE PROPAGATION

Bacteriophage propagation was performed as described by Sørensen et al. (2011) using a plate lysis method developed from Frost et al. (1999), Hansen et al. (2007). The propagating strains were grown overnight on blood agar Base II plates and harvested in BHI supplemented with 1 mM CaCl 2 and 10 mM MgSO 4 (CBHI). Bacterial suspensions were adjusted to an optical density of OD 600 = 0.35, incubated for 4 h at 37˚C under microaerobic conditions, mixed with phages at a multiplicity of infection (MOI) of 0.01 and incubated for 15 min at 37˚C to allow bacteriophage adsorption. Subsequently, 0.6 ml of the adsorbed suspensions were mixed with 5 ml of NZCYM overlay agar [NZCYM (Sigma) broth with 0.6% agar] at 45˚C and poured onto NZCYM 1.2% agar plates. After incubation, plates with lysis were flooded with sterile SM buffer and the phages eluted overnight with gentle shaking at 4˚C. Phage suspensions were harvested and filtered through a sterile 0.2 μm membrane filter.

BACTERIOPHAGE TITRATION

Bacteriophage titration was performed as previously described (Sørensen et al., 2011). Briefly, bacterial lawns were made by mixing 5 ml NZCYM overlay agar tempered at 45˚C with 0.5 ml bacteria harvested in CBHI from Base II agar plates and incubated for 4 h as described above and poured onto NZCYM plates. Phage solutions were spotted manually with three times 10 μl of 10 0 -10 −7 phage dilutions in SM buffer. Plates were incubated at 37˚C for 20-24 h under microaerobic conditions and the number of plaque-forming units per milliliter (PFU/ml) of undiluted phage suspensions was calculated. Usually, phage titers of 10 8 -10 9 PFU/ml could be obtained on both the propagating strains and C. jejuni MP21.

PLAQUE ASSAY

Susceptibility of a C. jejuni strain to the bacteriophages was performed using the plaque assay protocol described under Bacteriophage titration and determined as the efficiency of plaquing (EOP) in percent by dividing the PFU/ml on the test strain by the PFU/ml on the control strain (NCTC11168) multiplied by 100. The values represented are the mean count standard deviations derived from two to four independent experiments.

HIGH-RESOLUTION MAGIC ANGLE SPINNING NMR SPECTROSCOPY

High-resolution magic angle spinning (HR-MAS) NMR analysis of intact bacterial cells was performed as previously described (McNally et al., 2005).

IN VIVO EXPERIMENTS

One-day-old chickens were obtained from the Poultry Research Facility, Department of Agriculture, Food and Nutritional Science, University of Alberta. On the day of arrival, chickens were checked for C. jejuni colonization by cloacal swabs and divided in groups of four chickens. On day 2, chickens were first orally gavaged with either 10 7 F336 phages in SM buffer or with SM buffer alone. After approximately 30 min, the chickens were orally gavaged with a suspension containing 10 6 C. jejuni NCTC11168 in PBS or with PBS alone. Six days post infection, chickens were euthanized and the contents from one cecum were collected aseptically. The cecal contents were weighed and resuspended in phosphatebuffered saline at a final concentration of 0.1 g/ml. This suspension was subjected to 10-fold serial dilutions and plated onto Karmali selective agar plates to determine bacterial colonization levels.

One hundred microliters of the 10 −1 dilution was plated separately onto selective plates and all resulting bacterial colonies were pooled together. Half of this pool was used for sequencing and phage sensitivity and the other half was used for NMR analysis.

The experiment was repeated twice with consistent results. PCR of gene cj1421 was performed as previously described (Sørensen et al., 2011) while sequencing was done using the following primer: 1421-seq2-F (5 -CTCGAGTTATAAGGATATTTTAGATGAG-3 ). Gene cj1426 was PCR-amplified using the primers: 1426-PCR-F (5 -TTGAGAATTATGATAAGATGAAGG-3 ) and 1426-PCR-R (5 -TTTCCTAAGAATTCTTTACTTTCG-3 ) and then sequenced using the primers: 1426-seq-F (5 -AAGATCCAGATAAAAGAGAT TATTTGG-3 ) and 1426-seq-R (5 -ATCAGGAGAATCAAAAA TGATTTTTCC-3 ).

RESULTS

THE MeOPN MOIETY OF THE CPS OF C. jejuni NCTC11168 IS A RECEPTOR FOR SEVERAL PHAGES

We recently demonstrated that phage F336 requires the O-methyl phosphoramidate (MeOPN) moiety attached to the GalfNac of the C. jejuni NCTC11168 CPS for infection (Sørensen et al., 2011).

Frontiers in Cellular and Infection Microbiology www.frontiersin.org In our phage collection (Hansen et al., 2007) we found that five phages, including F336, rely on capsular moieties for infection of C. jejuni NCTC11168, since they do not form plaques on a kpsM capsule mutant (  , 2007). Our results showed that the attachment site of MeOPN in the CPS clearly influenced the efficiency of the phages to infect a particular strain (Figure 1). In general, all phages showed a higher EOP when MeOPN was situated on the GalfNAc residue (Figures 1A,E) as compared to MeOPN attached to the Hep residue in the CPS ( Figure 1C). A lower EOP obtained with MeOPN attached to the Hep residue was further confirmed by screening a cj1421 + cj1422 deletion strain complemented with cj1422 (McNally et al., 2007) with our five phages ( Figure 1F). Interestingly, the four phages F198, F287, F303, and F336 were not able to infect C. jejuni 11168H, which is the only strain expressing MeOPN on both GalfNAc and Hep (Figure 1B), suggesting that two MeOPN moieties may inhibit phage infection. Finally, we observed that the presence of two other phase variable CPS modifications, the 3-O-Me and 6-O-Me groups influenced the plaquing efficiency of the phages. We also isolated a 11168H single colony variant (11168H1) that by HR-MAS NMR and 1 H-31 P HSQC HR-MAS NMR analysis showed the same CPS profile as the NCTC11168 wild type strain (data not shown). The phage sensitivity profile observed with this strain (Figure 1G) confirmed the results obtained with NCTC11168 ( Figure 1A). Thus, the presence of the 6-O-Me group on the CPS reduced the plaquing efficiencies of phages F287 and F326 (Figures 1A,E), suggesting that other phase variable CPS modifications besides MeOPN affect phage infection.

C. jejuni PHAGES REQUIRE DIFFERENT COMBINATIONS OF PHASE VARIABLE CPS MODIFICATIONS FOR SUCCESSFUL INFECTION

Phase variable expression of the capsular modifications in C. jejuni NCTC11168 is well documented (Parkhill et al., 2000;St Michael et al., 2002;Szymanski et al., 2003). Three frozen stocks of NCTC11168 (MP24, MP25, and MP26) that had been passaged a number of times in our laboratory were analyzed by HR-MAS NMR and 1 H-31 P HSQC HR-MAS NMR (Figure 2). This analysis revealed differences in the four phase variable modifications of the CPS structure, i.e., the 3-O-Me, 6-O-Me, MeOPN-GalfNAc, and MeOPN-Hep as compared to our NCTC11168 wild type ( Table 4). We also included another NCTC11168 variant (V26) previously shown to have yet another different set of modifications (St Michael et al., 2002;Carrillo et al., 2004). Plaque assays using our five phages showed that these C. jejuni strains had distinct phage sensitivity profiles (Figures 3B-E). Interestingly, C. jejuni MP26 that expresses both MeOPN moieties, similar to the phage-resistant C. jejuni 11168H (Figure 1B), could be infected by four of the five phages although with different efficiencies ( Figure 3D). However, CPS of C. jejuni MP26 only contains the 3-O-Me group, whereas 11168H CPS contains both the 3-O-Me and 6-O-Me groups (Figures 1B and 3D). These results indicate that the presence of 6-O-Me may inhibit phage infection. This was further supported by the observation that none of the phages formed plaques when the 3-O-Me was exchanged with the 6-O-Me while retaining the MeOPN modification attached to GalfNAc (Figures 3A,E). Furthermore, the presence of the 6-O-Me group in combination with a MeOPN situated on the GalfNAc and a 3-O-Me group clearly inhibited infection by phage F287 and F326 (Figures 1E,G). Thus, our data suggest that the 6-O-Me group inhibits plaque formation in vitro and that all five phages require MeOPN attached to GalfNAc together with the 3-O-Me to obtain the most efficient infection. However, different combinations of the phase variable CPS modifications appear to influence binding of the phages differently. In particular, phage F326 showed the lowest EOP values ( Table 3; Figures 1 and 3) suggesting that other combinations of receptors, not investigated in this study, might be optimal for the binding of this phage.

SELECTION FOR PHAGE-RESISTANT VARIANTS DURING C. jejuni COLONIZATION OF CHICKENS

To assess whether C. jejuni is capable of becoming resistant to bacteriophage F336 in vivo during colonization of chickens, we orally gavaged 2-day-old chickens with F336 and subsequently with C. jejuni NCTC11168. After 6 days, the chickens were euthanized and the bacteria from the ceca were analyzed. We observed similar levels of colonization after 6 days of infection between the groups receiving bacteria only, as compared to those receiving bacteria and bacteriophages (i.e., 10 9 CFU/g cecal content, Table 5). The bacteria grown from the cecal contents were directly pooled from plates and analyzed by HR-MAS NMR. All bacteria obtained from chickens infected with C. jejuni alone showed an NMR profile that was the same as the NMR spectrum of the inoculum: the bacteria expressed one MeOPN attached to GalfNAc and the 3-O-Me group bound to Hep. However, bacteria from the group of chickens that received both bacteria and bacteriophages had lost the  MeOPN in all instances, except for one (Figure 4; Table 5). In chicken #238, infected with phage F336 and C. jejuni, the bacteria showed reduced levels of MeOPN present on both GalfNAc and Hep, but the bacteria also expressed high levels of 6-O-Me on the Hep (Figure 4).

To test whether the observed changes in CPS modifications resulted in phage resistance, we performed bacterial titration assays with all of the isolates recovered from the chickens. All isolates from the F336 infected birds had gained resistance to phage F336 and still showed similar levels of colonization (Table 5). Previously, we have shown that a loss of MeOPN on GalfNAc can be the result of a switch in the polyG region of the MeOPN transferase gene cj1421 (Sørensen et al., 2011). To determine whether the loss of MeOPN and subsequent phage resistance were the result of a switch in the polyG region, we sequenced gene cj1421 from the pooled colonies from both experiments. All phage-sensitive isolates showed 9 G residues in the polyG region, while all phageresistant isolates (except from chick 238) showed either 8 or 10 G residues in this region, which in both cases results in a nonfunctional transferase, due to a premature stop codon 1 or 5 amino acids after the polyG region, respectively (data not shown). Examination of the pooled colonies from chick 238 showed 9 G residues in the polyG region of cj1421, consistent with the observed MeOPN expression. However, sequencing of the polyG region of cj1426 from the pooled colonies from chick 238 showed 10 G residues indicating that the 6-O-Me transferase enzyme was functional (again consistent with the acquired expression of the Me modification in Figure 4) compared to 11 G residues in Frontiers in Cellular and Infection Microbiology www.frontiersin.org FIGURE 3 | Capsular polysaccharide structures expressed by NCTC11168 variants and the corresponding phage sensitivity profiles. The CPS structure is schematically illustrated (modified from Guerry and Szymanski, 2008). Phage sensitivity patterns are calculated as the 1 efficiency of plaquing (EOP) as compared to plaque formation on the NCTC11168 wild type strain. the parental strain lacking this modification. Thus, C. jejuni can become resistant to phage F336 during in vivo colonization of chickens by changing the expression of its CPS phase variable modifications.

DISCUSSION

Implementation of efficient phage therapies to target pathogenic bacteria requires detailed knowledge of the bacterial receptors recognized by the phages, as this bacteria-phage interaction is the first event in a process leading to lysis of the host organism. We previously identified the C. jejuni NCTC11168 CPS phase variable MeOPN modification as the receptor for phage F336 (Sørensen et al., 2011). The goal of the present study was to further explore the role of MeOPN and other C. jejuni phase variable CPS modifications, such as O-methylation in phage infection. We compared five different phages, including F336 that were unable to proliferate in an acapsular mutant of C. jejuni NCTC11168, and determined their ability to infect NCTC11168 variants containing the same CPS backbone sugars as NCTC11168, but with different modifications.

We found that the phages F198, F287, F303, F326, and F336 all infected C. jejuni most efficiently when the 3-O-Me modification on Hep and a MeOPN moiety was present, with higher EOPs observed when the MeOPN modification was attached to GalfNAc rather than Hep. Interestingly, when the 3-O-Me was exchanged with the 6-O-Me while retaining the MeOPN on GalfNAc, none of the phages formed plaques at all, demonstrating a clear role for the O-methyl Hep modifications for phage infection, in addition to the previously identified phage receptor, MeOPN (Sørensen et al., 2011). By looking at the predicted three dimensional (3D) structure of the C. jejuni NCTC11168 CPS (Guerry and Szymanski, 2008), it is possible that the presence or absence of one or more CPS modifications may lead to conformational changes that interfere with the phage binding process. Indeed it has been found that changes in the 3D conformation of the outer membrane protein OmpA, which serves as the receptor for many of the T-even phages infecting E. coli, leads to phage resistance (Riede and Eschbach, 1986). Furthermore, the spatial arrangement of the modifications may be important for phage recognition and some modifications may potentially block the phage binding site when Frontiers in Cellular and Infection Microbiology www.frontiersin.org they are expressed. Certainly masking the phage binding site is a well known resistance mechanism in other bacteria. One example is Staphylococcus aureus that masks its phage receptor with protein A, a cell-wall-anchored virulence factor for this bacterium (Nordstrom and Forsgren, 1974). In agreement with this hypothesis, we observed that when MeOPN was present on both the GalfNAc and Hep residues together with the 3-O-Me and 6-O-Me Hep modifications, then four of the five phages could no longer infect that particular C. jejuni 11168 strain. Thus, both the presence and absence of the capsular modifications appears to modulate phage infectivity of the NCTC1168 strain. Our results demonstrate that the phages infecting C. jejuni are highly specific in the recognition of the CPS and that each phage has evolved to recognize a particular combination of capsular modifications. One might speculate that the phages included in this study originated from the same ancestral phage that has co-evolved with C. jejuni to recognize different capsular surface structures by minor modifications in the phage receptor binding proteins (RBP). In support of this, sequencing of viral proteins from the C. jejuni phage CP220 revealed at least two distinct tail structures in the mature virions although propagated from a single plaque (Timms et al., 2010), suggesting a possibility for the phage to recognize multiple receptors. A similar phenomena has been observed for capsular phages infecting E. coli (Scholl et al., 2001). Indeed RBPs of other phages often have hypervariable regions in the receptor recognizing domains, reflecting the ability of the phage to easily accumulate mutations thereby changing the structure of the RBP to recognize modified receptors or new types of receptors, such as switching from protein to carbohydrate recognition (Montag et al., 1990;Drexler et al., 1991;Hashemolhosseini et al., 1994). The C. jejuni phages investigated in this study have similar genome sizes and morphologies and were previously isolated from duck and broiler intestines and duck abattoirs (Hansen et al., 2007). Although these phages all are able to infect the NCTC1168 strain, they do show different host ranges for C. jejuni strains of different Penner serotypes (Hansen et al., 2007). In addition, enzymatic restriction cleavage of the phage genomes revealed differences among the phages ( Table 2, Hansen et al., 2007). Thus, although a conserved head and tail architecture is maintained, it is apparent that genomic dissimilarities exist between the five phages compared in this study and this may account for the differences in host specificity.

In C. jejuni, the CPS are highly diverse, due to differences in the genetic compositions of the CPS loci, but also because many of the genes in these loci contain homopolymeric tracts making them prone to phase variation (Linton et al., 2001;St Michael et al., 2002;Szymanski et al., 2003;Karlyshev et al., 2005;McNally et al., 2005;Chen et al., 2008). Based on the number of phase variable modifications in the capsule of C. jejuni NCTC11168 alone, this may result in the expression of >700 CPS structural variants. Our results demonstrate that phase variable CPS surface structures clearly influence the phage attachments sites in C. jejuni NCTC11168. Phase variable expression also affects phage adsorption in Bordetella spp. These bacteria use phase variable expression of the BvgAS two-component regulatory system to alter their surfaces through the regulation of colonization and virulence factors and also expression of the phage BPP-1 receptor Prn (pertactin autotransporter; Liu et al., 2002). To combat these changes, the BPP-1 phage has a sophisticated mechanism of varying its RBP (∼10 +13 possible sequences) that approaches the levels of diversity observed for immunoglobulins (Liu et al., 2002;Doulatov et al., 2004), and the phage is thus able to overcome the resistance by an equally efficient mechanism. It was previously shown that CPS expression is important for C. jejuni colonization of the chicken gut as well as being important for interactions with human models of disease (Bacon et al., 2001;Jones et al., 2004). Here we show for the first time that the presence of phages selects for specific CPS variants in vivo in the chicken gut without altering the colonization levels, even though MeOPN expression influences C. jejuni invasion of human cells (van Alphen, unpublished results). Thus, the roles of phase variable CPS modifications, such as the MeOPN and the O-methyl groups, may not only be linked to adaptation to the human or chicken hosts, but may serve an important function for C. jejuni survival in the avian gut; an environment that contains a high number of bacteria as well as phages (Connerton et al., 2004;Hansen et al., 2007). Scott et al. (2007) also described that certain isolates of C. jejuni containing Mu-like prophage DNA sequences were capable of developing bacteriophage resistance in the chicken gut through a mechanism of reversible genomic rearrangement. Remarkably, these isolates inverted genomic segments up to 590 kb which also led to a decrease in chicken colonization and production of a functional Mu-like bacteriophage (Scott et al., 2007). The authors speculated that the development of phage resistance could be due to alterations in the bacterial surface receptor or through saturation of the receptor sites if the Mu phage is recognizing the same structures. Here, we have shown that phase variable modifications of the CPS influence phage infectivity, suggesting that the constant exposure of C. jejuni to phages in the avian gut selects for changes in the phase variable structures of the CPS leading to a continuing co-evolution of C. jejuni polysaccharides and phage RBPs. This should be considered when developing future strategies for phage therapy against C. jejuni in the chicken host and when determining the kinetics of resistance development and the timing for therapy administration. Since the alteration of bacterial surface receptors that reduces or eliminates phage adsorption to the host cell appears to be the most common mechanism of phage resistance (Bohannan and Lenski, 2000), continued investigations into the diversity of phage receptors in C. jejuni is required to select the most efficient combinations of phages for therapy.

Frontiers in Cellular and Infection Microbiologywww.frontiersin.orgFIGURE 1 | Capsular polysaccharide structures expressed by defined MeOPN transferase mutants and their affect on phage sensitivity. The CPS structure is schematically illustrated (modified from Guerry and Szymanski, 2008). Phage sensitivity patterns are calculated as the 1 efficiency of plaquing (EOP) as compared to plaque formation on the NCTC11168 wild type strain. (A) NCTC11168; (B) 11168H; (C) 11168HΔ1421; (D) 11168HΔ1421/Δ1422; (E) 11168HΔ1422; (F) 11168HΔ1421/Δ1422 + 1422; and (G) 11168H1. 2 Plaques were only detected in one out of three experiments. 3 MeOPN detected in lower amounts due to partial complementation. Abbreviations are: Galf NAc, N-acetylgalactosamine in the furanose configuration; MeOPN, O-methyl phosphoramidate; Hep, heptose; and O-Me, O-methyl.

in Cellular and Infection Microbiology www.frontiersin.org FIGURE 2 | High-resolution magic angle spinning NMR spectra showing the differences in CPS modifications between NCTC11168 variants MP24, MP25 and MP26. 1 H CPMG spectra of intact C. jejuni cells. Abbreviations are: Hep, D-glycero-α-L-gluco-Hep; Rib, β-D-Ribf ; GlcA, α-D-GlcpA6(NGro); Galf NAc, β-D-Galf NAc; MeOPN, O-methyl phosphoramidate; OMe, O-methyl; NAc, N-acetyl; TSP, trimethylsilyl propionic acid standard.

( A )
ANCTC11168; (B) NCTC11168 MP24; (C) NCTC11168 MP25; (D) NCTC11168 MP26; and (E) NCTC11168 V26. 2 MeOPN only detected in minor amounts. Abbreviations as above are: Galf NAc, N-acetylgalactosamine in the furanose configuration; MeOPN, O-methyl phosphoramidate; Hep, heptose; and O-Me, O-methyl.

Table 1 |
1Campylobacter jejuni strains.Serotype
Description
Reference

1447
HS:4c
Chicken isolate
Hansen et al. (2007)

NCTC12662
HS:5j
Origin not known
National Collection of

Type Cultures, UK

NCTC11168 (MP21)
HS:2
Wild type
Sørensen et al. (2011)

NCTC11168 MP24
HS:2
NCTC11168 variant passaged
This study

NCTC11168 MP25
HS:2
NCTC11168 variant passaged
This study

NCTC11168 MP26
HS:2
NCTC11168 variant passaged
This study

NCTC11168 V26
HS:2
NCTC11168 variant
Carrillo et al. (2004)

kpsM
Untypeable
NCTC11168 kpsM::kan does not have a capsule
Karlyshev et al. (2000)

11168R
Phage F336 resistant NCTC11168
Sørensen et al. (2011)

S11168R
Phage-sensitive 11168R
Sørensen et al. (2011)

11168H
HS:2
Hypermotile NCTC11168
Jones et al. (2004)

11168HΔ1421
Loss of MeOPN on Galf NAc
McNally et al. (2007)

11168HΔ1421/1422
Loss of MeOPN on Galf NAc
McNally et al. (2007)

Loss of MeOPN on Hep

11168HΔ1422
Loss of MeOPN on Hep
McNally et al. (2007)

11168HΔ1421/1422 + 1422
Loss of MeOPN on Galf NAc
McNally et al., 2007

Gain of MeOPN on Hep

11168H1
11168H variant
This study

Loss of 6-O-Me and MeOPN on Hep

Frontiers in Cellular and Infection Microbiology
www.frontiersin.org

Table 2 |
2Bacteriophages.Category a HhaI

restriction

pattern b

Origin
Reference

F198 III
c
Broiler intestine Hansen et al. (2007)

F287 III
d
Duck intestine
Hansen et al. (2007)

F303 III
d
Duck abattoir
Hansen et al. (2007)

F326 III
d
Duck intestine
Hansen et al. (2007)

F336 III
b
Duck intestine
Hansen et al. (2007)

a Category is based on genome size (all are 140 kb) as defined in

BACTERIOPHAGESBacteriophages used in this study are listed inTable 2. They all belong to the family of Myoviridae and have genome sizes of approximately 140 kb, but show different HhaI restriction patterns. Phages were propagated on C. jejuni NCTC12662, except for phage F336 where C. jejuni 1447 was usedb, 5 + 2 weak

bands; c, 4 bands; d, 3 bands.

with 5% calf blood (BA) or in brain heart infusion (BHI) broth
under microaerobic conditions (6% CO 2 , 6% O 2 , 84% N 2 , and
4% H 2 ) at 37˚C. For chicken experiments, bacteria were grown on
Karmali agar (Oxoid) supplemented with Campylobacter selec-
tive supplement under microaerobic conditions (10% CO 2 , 5%
O 2 , 85% N 2 ) at 37˚C.

Table 3 |
3Efficiency of plaquing (EOP) of phages related to F336 as compared to NCTC11168. a EOP is calculated in percent as the PFU/ml of the phages on the test strain divided by the PFU/ml obtained on NCTC11168 multiplied by 100.Phage
NCTC11168
11168R
S11168R
kpsM

F198
100 a
-b
82.5 ± 24.7
-

F287
100
0.04 ± 0.03
2300 ± 282.8
-

F303
100
-
350 ± 113.1
-

F326
100
0.02 ± 0.02
11.6 ± 6.7
-

F336
100
-
331.8 ± 64.3
-

b -No plaques formed.

Table 3 )
3. In our previous study, we isolated a phage F336 resistant NCTC11168 variant in vitro (11168R), which lost the MeOPN attached to the GalfNAc residue due to the presence of an additional G in gene cj1421 resulting in an off version of the gene(Sørensen et al., 2011). Here we found that 11168R had gained cross-resistance to all of the four phages: F198, F287, F303, and F326(Table 3). While phages F198 and F303 did not form plaques on C. jejuni 11168R at all, plaques were observed with F287 and F326, but with a much lower EOP compared to wild type NCTC11168. Hence, the MeOPN structure is important for infection by the four other phages, although two of these phages (F287 and F326) were able to infect a strain not expressing the MeOPN modification. Similar results were obtained when screening a cj1421 MeOPN transferase deletion mutant in the NCTC11168 wild type background (data not shown). Supporting this, the EOP of four of these phages, including F336, were fully restored to wild type NCTC11168 levels, while the EOP of phage F326 was partially restored, when we tested the phages against a 11168R revertant containing an intact cj1421 gene and returned expression of the CPS MeOPN (C. jejuni S11168R;Sørensen et al., 2011;Table 3). Thus, several phages rely on the MeOPN moiety attached to the GalfNAc residue of CPS for infection of C. jejuni NCTC11168.THE LOCATION OF THE MeOPN ON THE CPS IS IMPORTANT FOR PHAGE INFECTIONThe MeOPN moiety of C. jejuni NCTC11168 can be attached to
the GalfNAc and the Hep residues in the CPS (McNally et al.,
2007) and we speculated that the attachment site of MeOPN may
be important for phage infection. We therefore conducted plaque
assays and determined EOPs of the five phages F198, F287, F303,
F326, and F336 on the hypermotile NCTC11168 variant 11168H as
well as on defined mutants in the cj1421 and cj1422 MeOPN trans-
ferase genes in this background and compared to the NCTC11168
wild type strain. All of these strains have previously been character-
ized by HR-MAS NMR and 1 H-31 P heteronuclear single-quantum
correlation (HSQC) HR-MAS NMR for the presence/absence of
MeOPN in their CPS structure (McNally et al.

Table 4 |
4ppm Values for 1 H HR-MAS NMR spectra for NCTC11168 variants and MeOPN transferase status.C. jejuni strain
Hep a
Rib
GlcA
Galf NAc
3-O-Me
6-O-Me
NAc
MeOPN
cj1421/cj1422 (Galf NAc/Hep) b

NCTC11168 MP24
5.582
5.376
5.103
5.015
3.629
3.557
2.071
3.772
−/+

NCTC11168 MP25
5.599
5.363
5.148
5.039
-
3.569
2.066
-(3.772) c
−/−

NCTC11168 MP26
5.610
5.361
5.143
5.101
3.630
-
2.068
3.768/3.743
+/+

a Only anomeric proton resonances for carbohydrate residues are listed.

b Summary of sequencing results indicating the "on" (+) and "off" (−) status of the genes encoding the MeOPN transferases onto GalfNAc (cj1421) and Hep (cj1422).

c Only trace amounts of MeOPN detected.

Table 5 |
5Presence of MeOPN and phage sensitivity of C. jejuni isolated after colonization of chickens.Infected with
Bacterial counts (CFU/g)
MeOPN on Galf NAc
Phage sensitivity a

MP21 Inoculum
NR
NR
+
Yes

MP21 Karmali
NR
NR
+
Yes

Chick 405
MP21
5.7 × 10 9
+
Yes

Chick 406
MP21
4.1 × 10 9
+
Yes

Chick 407
MP21
9.9 × 10 8
+
Yes

Chick 408
MP21
1.1 × 10 9
+
Yes

Chick 505
MP21 + F336
7.9 × 10 8
−
No

Chick 506
MP21 + F336
5.0 × 10 9
−
No

Chick 507
MP21 + F336
5.8 × 10 9
−
No

Chick 508
MP21 + F336
8.5 × 10 8
−
No

Chick 343
MP21
2.0 × 10 9
+
Yes

Chick 344
MP21
2.1 × 10 9
+
Yes

Chick 345
MP21
1.6 × 10 9
+
Yes

Chick 346
MP21
3.6 × 10 9
+
Yes

Chick 237
MP21 + F336
1.2 × 10 9
−
No

Chick 238
MP21 + F336
5.5 × 10 9
+ b
No

Chick 239
MP21 + F336
4.7 × 10 9
− c
No

Chick 240
MP21 + F336
4.1 × 10 9
−
No

Chicks 405-408 and 505-508 are from the first experiment, while chicks 343-346 and 237-240 are from the second experiment.

a

Yes: plaques formed on isolated strain, No: no plaques formed on isolated strain.

b Bacteria isolated from this chick showed reduced MeOPN levels (of both MeOPN groups) and enhanced levels of 6-O-Me.

c MeOPN levels barely above the baseline could be detected from bacteria isolated from this chick.

NR, not relevant.

Frontiers in Cellular andInfection Microbiology www.frontiersin.org FIGURE 4 | NMR analysis of intact C. jejuni cells recovered from chicken cecal contents. (A) 1 H HR-MAS NMR CPMG spectra, displaying the capsular profile and (B) corresponding 1D 1 H-31 P HSQC spectra specifically demonstrating the phosphoramidate resonances. Black arrowhead: note the change in the Galf NAc resonance due to the absence of MeOPN. White arrowhead: note the absence of the MeOPN specific resonance. in Cellular and Infection Microbiology www.frontiersin.orgFrontiers
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ACKNOWLEDGMENTSWe are grateful to B. Wren for the gift of the C. jejuni NCTC11168 kpsM mutant. We sincerely appreciate the expert technical assis-Conflict of Interest Statement:The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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