Detection of Carbapenem Resistance in Salmonella Species from a Tertiary Hospital in Eastern Cape, South Africa

Aims: Broad-spectrum carbapenem group is the current therapy for strains of Enterobacteriaceae that express extended spectrum beta-lactamases (ESBLs). However, recent reports of therapeutic failures of carbapenems with strains that produce multiple β -lactamases are being documented. This study profiled antibiotic resistance in clinical isolates of Salmonella species in a tertiary hospital in the Eastern Cape of South Africa with the aim of identifying the status of Salmonella therapy in the region. Study Design: This is an analytical study. Salmonella isolates (119) from 96 blood and 23 stool specimens of patients attending Nelson Mandela Academic Hospital Complex (NMAHC) and surrounding clinics, Eastern Cape, South Africa collected for surveillance purposes over a period of 3 years (2006 – 2009) were obtained from National Institute of Communicable Diseases, (NICD), for analysis between 2010 and 2011. Methodology: Preliminary identification and serotyping were done at the NICD. The identification and antimicrobial susceptibility profile of isolates were confirmed with an Autoscan-4 antimicrobial susceptibility system. The MIC of ertapenem and imipenem tested for all Salmonella spp. were ≥ 2 mg/L and ≥ 4 mg/L respectively. Results: A considerable portion of the isolates 59/119 (49.6%) showed pentavalent resistance to some antibiotics including ampicillin and amoxicillin. Of the 59 multiply resistant isolates, 14 (23.7%) were resistant to 1 or more of the carbapenems examined. The phenotypic determination of ESBLs resulted in 25 (21.0%) ESBL-positive Salmonella isolates. Using Fisher's exact test, the proportion of carbapenem resistance isolates was significant at P value 0.032. Conclusion: The growing resistance of Salmonella isolates to carbapenem drugs in this setting call for caution in usage since this is a pointer to fewer options in the choice of drugs for ESBL’s therapy. Contact precautions should be put in place to forestall further transmission.


INTRODUCTION
Antibiotics are often not recommended in the treatment of enteritis. It is believed that antibiotics prolong the carrier state of enteritis due to Salmonella. Nevertheless, systemic Salmonella infections and infections in the vulnerable groups qualify for antibiotic therapy [1]. These infections can be treated with ampicillin, gentamicin, trimethoprim/ sulfamethoxazole, ceftriaxone, amoxicillin, or ciprofloxacin [2]. However, some Salmonella strains like other bacteria in Enterobacteriaceae have become resistant not only to commonly used antibiotics but exhibit multidrug resistance. The resistance mechanism may be in the form of β-lactamase production or alterations in penicillin-binding proteins (PBPs) [3]. The emergence and spread of extended-spectrum βlactamases (ESBLs) -production among isolates of Enterobacteriaceae both from community and health-care settings have engendered fear. Broad-spectrum carbapenem group is the last resort therapy for strains of Enterobacteriaceae that express extended spectrum beta-lactamases [4].
Carbapenems and penems are known to possess high potency of antimicrobial activity against a broad spectrum of bacteria [4] and were known to be β-lactamase resistant not readily hydrolyzed by almost all β-lactamases, but undergo metabolism by β-lactamases of mammalian origin known as dehydropeptidases (DHPs) [5]. Carbapenems (doripenem, ertapenem, imipenem, meropenem) like all other β-lactam antibiotics (penicillins, cephalosporins, carbacephems and monobactams) have the same bactericidal mechanism of action; blocking a critical step in bacterial wall synthesis [6]. However, this currently most successful class of antibiotics is showing signs of vulnerability with recent reports of therapeutic failures of carbapenems with strains that produce multiple β-lactamases [7]. These bacterial strains may carry genes encoding β-lactamases that confer resistance to broad-spectrum β-lactams, including carbapenems.
The ESBL enzymes that confer resistance to extended spectrum cephalosporin and carbapenem antibiotics have over the years developed into what are known as carbapenemases. The enzymes involved in the hydrolysis of carbapenems are serine carbapenemases of the Klebsiella pneumoniae carbapenemase (KPC) type, of the New Delhi metallo-β-lactamase (NDM) or Verona integronencoded metallo-β-lactamase (VIM) types and the imipenemase (IMP)metallo-β-lactamase [8]. Carbapenemase resistance was also reported to have developed during ertapenem treatment of ceftriaxone-resistant and ciprofloxacin-resistant Salmonella enterica serotype Typhimurium [9]. Hence, Salmonella has been described as being very plastic in developing antimicrobial resistance [9]. The resistance determinants for carbapenem frequently co-exist in mobile genetic elements (plasmids) with resistance determinants (chromosome-encoded) to other antibiotics that are commonly used against Enterobacteriaceae infections leaving few therapeutic options available [10][11]. Carbarpenem resistant organisms are to be recognized as epidemiologically important and an understanding of the prevalence in their region crucial in controlling transmission [8 study profiled carbapenem antibiotic resistance in clinical isolates of Salmonella tertiary hospital in the Eastern Cape province of South Africa with the aim of identifying the current state of Salmonella therapy in the region.

Study Design and Sampling
This is an analytical study in which isolates (119) from 96 blood and 23 stool specimens of patients attending Nelson Mandela Academic Hospital Complex (NMAHC) and surrounding clinics collected for surveillance purposes and deposited at the repository of the National Institute of Communicable Diseases, NICD, Johannesburg, South Africa over a period of 3 years (2006 -2009) were collected from the Centre for this study.

Bacterial Isolates Characterization
The isolates which were previously identified and serotyped at the NICD were first subcultured for purity on blood agar and Tryptic Soy Agar and incubated at 37°C for 18 -24h. Preliminary morphological identification was done by plating on MacConkey Agar plates incubated at 37 18 -24h and Gram reaction. The identity and the antibiotic susceptibility pattern of the pure culture of the isolates were subsequently confirmed using the Microscan System (Siemens Behring, South Africa). The Gram Negative Combo 50 Panel (Siemens-Dade Behring, South Africa) was used for the simultaneous 3 region crucial in controlling transmission [8]. This ofiled carbapenem antibiotic resistance species in a tertiary hospital in the Eastern Cape province of with the aim of identifying the therapy in the region.

Design and Sampling
This is an analytical study in which Salmonella 96 blood and 23 stool specimens of patients attending Nelson Mandela Academic Hospital Complex (NMAHC) and collected for surveillance deposited at the repository of the National Institute of Communicable Diseases, NICD, Johannesburg, South Africa over a period 2009) were collected from the and The isolates which were previously identified and serotyped at the NICD were first subcultured for purity on blood agar and Tryptic Soy Agar and 24h. Preliminary one by plating on MacConkey Agar plates incubated at 37°C for 24h and Gram reaction. The identity and the antibiotic susceptibility pattern of the pure culture of the isolates were subsequently confirmed System (Siemens-Dade Behring, South Africa). The Gram Negative Dade Behring, South was used for the simultaneous determination of minimum inhibitory concentration of the antibiotics and ESBLs phenotypes of the isolates according the guide of manufacturer. The Combo 50 panels contain ertapenem and imipenem at concentrations mg/L and ≥4 mg/L respectively. Other types of antibiotics on the panels had concentrations (mg/L) ranged between 0.5 and 64. The reference method was microaccording to Clinical laboratory Standards Institute guidelines for Gram negative bacteria [12].

Statistical Analysis
The results of susceptibility tests were subjected to statistical analysis using Fisher's exact test of independence while descriptive analysis was done with SPSS version 18.0 (South Africa) level of significance was set at P =

RESULTS AND DISCUSSION
The subcultured isolates were identified as Gram negative non-lactose fermenting rods and confirmed to be Salmonella species with various serovars: S. enterica serovar Typhi being the highest 69/119 (57.9%) followed by serovar Typhimurium 28/119 (23.5 distribution of these and other serovars shown (Fig. 1). Most isolates were resistant to amoxicillin, ampicillin, trimethoprim/ sulfamethoxazole and tetracycline with reduced susceptibility to ciprofloxacin. Resistance to five or more CLSI antibiotics subclasses was detected in 59/119 (49.6%) of the isolates.

SSION
The subcultured isolates were identified as Gram lactose fermenting rods and species with various serovar Typhi being the %) followed by S. enterica serovar Typhimurium 28/119 (23.5%), the distribution of these and other serovars is as shown (Fig. 1) Table 1). This finding is consistent with the similarity in high burden of MDR strains with increasing resistance to quinolones and thirdgeneration cephalosporins reported by Ke et al. [13].
A high frequency of resistance to drugs such as ampicillin, tetracycline and trimethoprim/ sulfamethoxazole was also observed in this study. However, the worrisome observation is the growing resistance to carbapenems. The proportion of isolates resistant to carbapenem group was significant at P value .03. Increasing isolation of Gram negative bacteria resistant to carbapenem has been strongly correlated to usage of the drug in therapy [14]. Resistance to carbapenem drugs is mediated by mobile carbapenemase located on plasmids [11]. This mobile elements carriage of resistant determinants with the genetic plasticity of the Enterobacteriaceae, has reportedly led to rampant intra and interspecies transfer of these elements and emergence of organisms with resistance to virtually all antibiotics [15].
The widespread dissemination of different variants of carbapenemase has been predicted.
There has been report of the spread of KPC-2 carbapenemases among Klebsiella pneumoniae, E. coli, Salmonella spp. and Enterobacter spp. in the USA [16], KPC-2-and KPC-3-producing K. pneumoniae in Israel [17], KPC-2 and IMP-4 in China [18]. The newest carbapenemase to emerge is New Delhi Metallo beta-lactamase 1 (NDM-1) discovered in Sweden from a patient previously hospitalized in India [10]. A proposed regimen to slow the development of resistance to the current armamentarium is avoiding prolonged antibiotic use or under-dosing, using pharmacokinetic and pharmacodynamic principles to choose dosing regimens, and encouraging early and aggressive empirical therapy, followed by de-escalation and narrowing the antimicrobial spectrum when culture results become available [7]. Combination therapy of rifampicin with doripenem and colistin or double carbapenem with ertapenem has been suggested [20], while fosfomycin as part of combination regimens might be useful as a lastresort option [22]. The relative safety of Carbapenems is a real advantage over the concern of selection of carbapenem resistant isolates, thus there is a need to continue development of the compounds [23].

CONCLUSION
This study reports the detection of carbapenem resistance in clinical isolates of Salmonella in the Eastern Cape region of South Africa. The study showed that the emergence of carbapenemresistant Salmonella has serious implications in such a resource-limited hospital in sub-Saharan Africa. Given the lack of new drugs to treat these infections, efforts should be focused on infection control practices among health-care personnel, visitors and patients such as contact precautions which include patients isolation, adherence to recommendations for gown and glove use by health-care workers, proper hand hygiene before and after patient contact, before oral intake of drugs, food and drinks and environmental sanitation.